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Asymmetric dimethylarginine (ADMA) levels are increased in patients with fibromyalgia: Correlation with tumor necrosis factor-α (TNF-α) and 8-iso-prostaglandin F2α (8-iso-PGF2α)
Clinical Biochemistry 44 (2011) 364–367
Contents lists available at ScienceDirect
Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m
Asymmetric dimethylarginine (ADMA) levels are increased in patients with fibromyalgia: Correlation with tumor necrosis factor-α (TNF-α) and 8-iso-prostaglandin F2α (8-iso-PGF2α) G. Topal a,⁎, A. Donmez b, B.S. Uydes Doğan a, M. Kucur c, D. Taspinar Cengiz d, F.B. Berkoz b, N. Erdogan b a
Department of Pharmacology, Istanbul University, Faculty of Pharmacy, 34116, Beyazit, Istanbul, Turkey Department of Medical Ecology and Hydroclimatology, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey c Fikret Biyal Central Biochemistry Laboratory, Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey d Department of Statistics, Istanbul Commerce University, Faculty of Arts and Sciences, Istanbul, Turkey b
a r t i c l e
i n f o
Article history: Received 8 October 2010 Received in revised form 24 December 2010 Accepted 22 January 2011 Available online 1 February 2011 Keywords: Fibromyalgia ADMA TNF-α IL-6 8-iso-PGF2α
a b s t r a c t Objective: The aim of the study was to investigate serum levels of asymmetric dimethylarginine (ADMA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and plasma levels of 8-iso-prostaglandin F2α (8-iso-PGF2α) in patients with fibromyalgia. Design and method: Twenty-seven patients with fibromyalgia and twenty healthy controls were enrolled in this study. ADMA, TNF-α, IL-6 and 8-iso-PGF2α levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of ADMA and TNF-α and plasma levels 8-iso-PGF2α were significantly increased in patients with fibromyalgia compared to controls. However, no significant difference was observed in IL-6 levels between the two groups. ADMA concentrations were positively correlated with TNF-α and 8-iso-PGF2α levels in patients with fibromyalgia. Conclusion: This is the first study reporting that ADMA levels are significantly elevated in patients with fibromyalgia in association with increased 8-iso-PGF2α and TNF-α concentrations. Thereby, ADMA could be suggested as a reliable marker of endothelial dysfunction in patients with fibromyalgia. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Fibromyalgia (FM) is a form of nonarticular rheumatism characterized by chronic wide-spread pain, tender points, fatigue, nonrefreshing sleep and morning stiffness. The American College of Rheumatology 1990 Fibromyalgia Classification Criteria are based on a history of chronic widespread musculoskeletal pain for at least 3 months and more than 11 of 18 painful tender points with palpation [1]. FM is a very common rheumatic disorder among women, with a prevalence of 3.6% in Turkish population [2] which is similar to the prevalence of USA [3]. The etiology of FM is still unknown and the pathogenesis is not clearly understood. Of interest, an overlap is pointed out between FM and depression where a relationship between depression and coronary heart disease is very common. Thus, it is reasonable to propose a correlation between FM and coronary heart disease. In relation, an increase in the level of tenderness and occurrence of FM symptoms among patients with pathological findings on coronary angiography was documented [4]. In another study, left ventricular (LV) dysfunction was observed in FM
⁎ Corresponding author. Fax: +90 212 527 18 25. E-mail address: [email protected] (G. Topal).
patients with a high Fibromyalgia Impact Questionnaire (FIQ) score [5]. Hereby, the need of further investigations focusing on vascular and endothelial functions was signified in patients with FM. Favorably, endothelial dysfunction was reported very recently in patients with FM as assessed by the reduction in flow mediated dilation (FMD) of brachial artery [6]. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) and thus presumed as a marker of endothelial dysfunction. Elevated ADMA concentration has been recognized as an independent risk factor in atherosclerosis and related cardiovascular diseases [7]. Moreover, increased ADMA concentrations have been reported in various inflammatory rheumatic diseases, where endothelial dysfunction has been defined, including rheumatoid arthritis [8], ankylosing spondylitis [9] and Behçet's disease [10]. However, to the best of our knowledge, no information is evident in literature so far in relation to an association between ADMA levels and FM. It has previously been suggested that oxidative stress and proinflammatory cytokines play a significant role in the pathogenesis of FM [11,12]. Inflammation and oxidative stress are important determinants of the endothelial dysfunction by several mechanisms including the deterioration of nitric oxide (NO) bioavailability [7]. In relation, decreased NO production was suggested in FM in a previous
0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.01.011
G. Topal et al. / Clinical Biochemistry 44 (2011) 364–367
study [13]. Of note, an association between oxidative stress, proinflammatory cytokines and ADMA levels is demonstrated in various pathologies [14–16], whereas, no data are available in FM. Therefore, in this study, we aimed to examine serum levels of ADMA, a reliable marker of endothelial dysfunction, the proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as plasma levels of 8-iso-PGF2α, one of the important indicator of oxidative stress and lipid peroxidation, in patients with FM comparatively with control subjects. This study was also designed to examine the correlations among ADMA, TNF-α, IL-6 and 8-isoPGF2α levels in patients with FM. Patients and methods Patients
Table 2 Clinical characteristics of the patients with FM and controls.
Total cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL) Triglycerides (mg/dL) FIQ total Pain Fatigue Morning stiffness Anxiety Depression Tender points BDI Disease duration (year)
FM patients (n = 27)
Controls (19)
p-value
195.40 ± 33.35 51.67 ± 11.57 104.40 ± 17.73 100.80 ± 25.18 55.44 ± 15.98 5.88 ± 2.10 6.56 ± 2.36 4.60 ± 3.00 6.28 ± 2.85 4.72 ± 2.76 16.42 ± 1.77 10.76 ± 7.74 9.28 ± 5.06
184.4 ± 23.68 51.23 ± 11.26 117.4 ± 29.22 101.7 ± 42.69 27.57 ± 7.72 1.68 ± 1,24 3.05 ± 1.64 0.10 ± 0.31 2.78 ± 1.43 2.47 ± 1.71 2.11 ± 1.24 3.52 ± 2.91 –
0.326 0.925 0.198 0.951 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001
HDL: high-density lipoprotein; LDL: low-density lipoprotein; FIQ: fibromyalgia impact questionnaire; BDI: Beck depression inventory. Values are given as mean ± SD.
Patients with FM were recruited from the musculoskeletal disorders outpatient clinic of Department of Medical Ecology and Hydroclimatology, Istanbul Medical Faculty. The study was performed on 27 patients satisfying the American College of Rheumatology Criteria for the classification of FM and 20 healthy controls matched for age. One healthy volunteer from control group recruited the study without any excuse. The patient and control groups were consisted of subjects at ages between 20 and 60, who did not have any other conditions in terms of chronic diseases and drug usage that may influence laboratory evaluation. The sociodemographic characteristics of the study groups are documented in Table 1. Laboratory tests used for the evaluation of patients and controls included complete blood count, erythrocyte sedimentation rate, glycaemia, serum lipid levels, hepatic and renal function tests, thyroid stimulant hormone level and X-ray. Exclusion criteria included the presence of any inflammatory autoimmune diseases, diabetes, renal disease, liver disease, known heart disease and pregnancy and use of any non-steroidal antiinflammatory drugs (NSAIDs) during a 15 days period before the collection of blood samples. Furthermore, our patients were allowed to use acetaminophen (max. 1 g/day) as an analgesic only when it is appropriate. The Institutional Review Board of the Istanbul University Faculty of Medicine approved the study plan and all patients and healthy controls voluntarily participated in the study and gave their written informed consent. Clinical evaluation FIQ was used for symptom measures and global evaluation of the patients. FIQ is a well-documented and widely-used measure in FM. FIQ is a ten-item questionnaire that measures physical functioning, work status, depression, anxiety, sleep, pain, stiffness, fatigue and well-being. [17]. FIQ is a widely recommended tool in FM research. The Turkish translation of the FIQ was found to be valid [18]. Biochemical analysis Venous blood was collected from each participant into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) or no anticoagulant. Immediately after delivery, serum or plasma was
Table 1 Sociodemographic characteristics of the study population.
Age (year) Sex (F/M) BMI (kg/m2) Education duration (year)
365
FM patients (n = 27)
Controls (19)
p-value
40.72 ± 10.58 23/2 24.52 ± 4.87 11.60 ± 4.74
40.00 ± 8.80 19/4 24.00 ± 3.18 14.68 ± 2.42
0.812 0.210 0.685 0.013
BMI: body mass index, F/M: female/male; values are given as mean ± SD.
isolated by centrifugation at 2500g for 10 min at 4 °C and stored at −80 °C until the analyses were performed. Plasma 8-iso-PGF2α levels and serum TNF-α, IL-6 and ADMA levels were analyzed by using commercial enzyme-linked immunosorbent assay (ELISA) kit for 8-iso-PGF2α, TNF-α, IL-6 (Cayman Chemical, Ann Arbor, MI, USA) and for ADMA (DLD, Diagnostika GmbH, Hamburg, Germany) according to the manufacturer's instructions. Intra-assay coefficient of variation was 5.7%, 10.6%, 15.4%, 14.9% and inter-assay coefficient of variation was 10.3%, 18.3%, 10.4% and 23.1% for ADMA, 8-isoPGF2α, TNF-α and IL-6, respectively. All samples were assayed in duplicate and the concentration was calculated from a standard curve of the corresponding recombinant proteins. Serum lipid levels were measured according to standard procedures by the local clinical laboratory. Statistical analysis All statistical analyses were performed using Statistical Package for Social Sciences software (SPSS, Chicago, IL, USA) version 18.0 for windows “n” denotes the number of FM and control subjects. All values are given as mean ± standard deviation (SD). Differences between the FM and control groups were evaluated by using Student's unpaired t-test. For determination of correlation between results, the Pearson's correlation coefficient was performed. Furthermore, the partial correlation analyses were fitted to evaluate the extend to which the associations between variables was altered after adjusting for age, gender and body mass index (BMI). p b 0.05 was considered statistically significant. Results Table 1 presents the sociodemographic characteristics of the study population. No significant differences were found between the groups with regard to age, gender and BMI. But, the controls had longer education duration than patients with FM. Clinical characteristics of the patients and control subjects are presented on Table 2. The main symptoms of FM are found to be pain, fatigue, and morning stiffness. Anxiety and depression are also frequently observed in patients with FM. In addition, no significant difference was determined in the lipid profile of FM and control groups (Table 2). Serum ADMA (p = 0.039) and TNF-α (p = 0.008) levels and plasma 8-iso-PGF2α levels (p = 0.001) were significantly increased in patients with FM compared to controls (Table 3). However, no significant difference was observed in terms of serum IL-6 levels between two groups (p = 0.992). According to the results of unadjusted correlation analysis, ADMA concentrations were found to be correlated significantly with both TNF-α and 8-iso-PGF2α levels (r = 0.549, p = 0.007 and r = 0.463,
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G. Topal et al. / Clinical Biochemistry 44 (2011) 364–367
Table 3 Biochemical parameters in patients with FM and controls.
ADMA (μmol/L) TNF-α (pg/mL) IL-6 (pg/mL) 8-iso-PGF2α (pg/mL)
FM patients (n = 27)
Controls (n = 19)
p-value
0.78 ± 0.31 21.37 ± 15.35 10.33 ± 8.55 165.37 ± 98.71
0.58 ± 0.30 10.32 ± 5.86 10.29 ± 11.47 72.78 ± 59.21
0.039 0.008 0.992 0.001
Values are mean ± SD.
Table 5 Adjusted correlation of ADMA versus TNF-α, 8-iso-PGF2α and IL-6 levels in patients with FM and controls.
TNF-α 8-iso-PGF2α IL-6
FM patients (27)
Control (19)
r
p
r
p
0.613 0.612 0.495
0.005 0.004 0.061
−0.030 0.167 −0.057
0.916 0.538 0.853
Partial correlation analyses were used to adjust for age, gender and BMI.
p = 0.015, respectively) in patients with FM (Table 4). After adjustment for age, gender and BMI with partial correlation, the observed associations between these variables remained significant (Table 5). In addition, no significant correlation was observed between ADMA and IL-6 concentrations in both unadjusted and adjusted correlation analysis in patients with FM. Whereas, in control subjects, no significative correlation was observed between the variables in either the adjusted or unadjusted analyses (Tables 4 and 5). Discussion In this study, we demonstrated that serum ADMA and TNF-α levels and plasma 8-iso-PGF2α levels were significantly elevated in patients with FM while IL-6 levels were unaltered. Moreover, ADMA levels were significantly correlated with TNF-α and 8-iso-PGF2α levels in these patients. ADMA, an endogenous inhibitor of NO synthase (NOS), is a relevant and independent risk factor for cardiovascular diseases. Endothelial dysfunction is commonly observed in cardiovascular diseases which is mainly attributed to oxidative stress, dyslipidemia, genetic factors and other causes. There is a growing evidence indicating that increased levels of ADMA may also contribute to endothelial dysfunction [7,19]. Moreover, recent studies suggest that ADMA is also a potential candidate to enhance vascular complications in patients with inflammatory rheumatic diseases including rheumatoid arthritis [8], ankylosing spondylitis [9] and Behçet's disease [10]. In the current study, we have reported for the first time that serum ADMA levels are elevated in patients with FM which may reflect the presence of endothelial dysfunction. Indeed, our findings presented an explanation for the previous hypothesis of endothelial dysfunction and decrease of NO production in FM [13]. Thus, enhanced ADMA generation might be associated with the risk of future cardiovascular events in these patients. Of interest, an association between FM and coronary heart diseases has been emphasized in the recent studies. Firstly, Ablin J.N. et al. demonstrated an increase in the levels of tenderness and in occurrence of FM symptoms among patients with pathological findings on coronary angiography [4]. Then, in another study, left ventricular (LV) dysfunction was observed in FM patients with a high FIQ score [5]. Finally, in a latest study, decreased FMD of branchial artery was reported in patients with FM which supports the role of endothelial dysfunction [6]. Hence, in the present study, increased ADMA levels were defined in patients with FM which provide a further evidence in the relationship between endothelial dysfunction, cardiovascular disorders, and FM. However, additional research is required to demonstrate the association between ADMA levels and flow-mediated vasodilation directly in patients with FM.
Table 4 Correlation of ADMA versus TNF-α, 8-iso-PGF2α and IL-6 levels in patients with FM and controls. FM patients (27)
TNF-α 8-iso-PGF2α IL-6
Control (19)
r Pearson
p
r Pearson
p
0.549 0.463 0.126
0.007 0.015 0.607
0.049 0.153 0.220
0.857 0.546 0.431
Proinflammatory cytokines might play a regulatory role in ADMA metabolism. In an in vitro study carried on human umbilical vein endothelial cells, TNF-α was shown to increase the accumulation of ADMA whereas decreased dimethylarginine dimethyl-aminohydrolase (DDAH) activity which is responsible for the metabolism of ADMA [20]. Furthermore, in patients with acute liver failure, elevated plasma concentration of ADMA was shown to correlate with the concentrations of TNF-α and IL-6 possibly through the regulation of ADMA metabolism by these cytokines [16]. The activation and regulation of pro-inflammatory cytokine pattern are implied in a variety of disease states such as, rheumatoid arthritis, ankylosing spondylitis [21]. Moreover, the association between FM and proinflammatory cytokines has been evaluated in some of recent studies [11,22]. In support, in the current study, we have found elevated serum levels of TNF-α. However, the other proinflammatory cytokine IL-6 levels were found indifferent in patients with FM compared to controls. These data are consistent with other studies in the literature reporting similarity in serum or plasma levels of IL-6 [11,23,24] whereas an increase in the levels of TNF-α [11,22] in patients with FM compared to healthy controls. Lastly, our results indicating a positive correlation between increased ADMA and TNF-α levels in patients with FM, which was significant even after adjustments for age, gender and BMI, supported the hypothesis of an association between a cytokinedriven inflammatory response and ADMA levels. F2-isoprostanes including 8-iso-PGF2α are currently considered the best available biomarkers of oxidative stress and lipid peroxidation in vivo [25]. Elevated 8-iso-PGF2α levels have been found in different inflammatory conditions including rheumatic diseases as well [26]. Additionally, increased levels of plasma 8-iso-PGF2α have been demonstrated in chronic fatigue syndrome which displays similar symptoms with FM [27]. In our study, it is clearly evident that plasma 8-iso-PGF2α levels are significantly higher in patients with FM than controls, indicating an enhanced oxidative stress and lipid peroxidation in these patients. Actually, this finding is in agreement with previous studies that have linked oxidative stress and lipid peroxidation to FM pathogenesis [12,28–31]. In fact, this is the first study indicating the role of 8-iso-PGF2α, possibly as an oxidant candidate, in FM. In contrary to our findings, Chung et al. did not demonstrate an alteration in urinary excretion of 8-iso-PGF2α in FM patients [32]. This discrepancy may be related to the differences in test samples or the methodology used in the analysis of 8-iso-PGF2α. Oxidative stress is now considered as a crucial factor in ADMA accumulation [33]. In relation, the increased ADMA generation associated with 8-iso-PGF2α levels was very recently reported in inflammatory bowel diseases [14]. Hence, in the present study, this relevance is furtherly supported by the positive correlation verified between ADMA and 8-iso-PGF2α levels in patients with FM which remained significant even after adjustments for age, gender and BMI. This study had some limitations that should be considered. Firstly, the number of patients is limited and therefore, this issue requires further studies with much larger subject population to give greater confidence in statistical correlations. Secondly, we did not assess vascular endothelial function in patients with FM. Further studies such as the measurement of flow mediated vasodilation are
G. Topal et al. / Clinical Biochemistry 44 (2011) 364–367
appropriate to validate the correlation between increased ADMA levels and endothelial dysfunction in FM. In conclusion, present study primarily shows that in patients with FM an enhancement is prominent in serum ADMA levels which is positively correlated with increased 8-iso-PGF2α and TNF-α concentrations. These results suggest that endothelial dysfunction might play a prominent role in the pathophysiology of FM. In consequence, patients with FM might be more prone to cardiovascular disorders. Hence, current findings strengthen the earlier hypothesis of Ablin et al. [4] and Cho et al. [5] on the association between coronary heart diseases and FM as well as the latest report by Cho et al. [6] on the presence of endothelial dysfunction assessed by a decrease in FMD of branchial artery in patients with FM. Lastly, our study strongly indicates the necessity for further investigations that reveal vascular functional properties particularly in the follow up period in patients with FM. Conflict of interest The authors declare that they have no competing interests. Acknowledgments The authors thank Ms. Nevin Bezgal and Ms. Nuray Çetin for excellent technical assistance. References [1] Wolfe F, Smythe HA, Yunus MB, Bennett RM, Bombardier C, Goldenberg DL, et al. The American College of Rheumatology 1990 Criteria for the classification of fibromyalgia. Report of the Multicenter Criteria Committee. Arthritis Rheum 1990;33:160–72. [2] Topbas M, Cakirbay H, Gulec H, Akgol E, Ak I, Can G. The prevalence of fibromyalgia in women aged 20–64 in Turkey. Scand J Rheumatol 2005;34:140–4. [3] Wolfe F, Ross K, Anderson J, Russell IJ, Hebert L. The prevalence and characteristics of fibromyalgia in the general population. Arthritis Rheum 1995;38:19–28. [4] Ablin JN, Beilinson N, Aloush V, Elkayam O, Finkelstein A. Association between fibromyalgia and coronary heart disease and coronary catheterization. Clin Cardiol 2009;32(6):E7–E11. [5] Cho KI, Lee JH, Lee HG, Kim SM, Kim TI. Assessment of myocardial function in patients with fibromyalgia and relationship to chronic emotional and physical stress. Korean Circ J 2010;40:74–80. [6] Cho KI, Lee JH, Kim SM, Lee HG, Kim TI. Assessment of endothelial function in patients with fibromyalgia-cardiac ultrasound study. Clin Rheumatol 2010 Oct 19. [Epub ahead of print]. [7] Boger RH. Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond. Ann Med 2006;38:126–36. [8] Surdacki A, Martens-Lobenhoffer J, Wloch A, Marewicz E, Rakowski T, WieczorekSurdacka E, et al. Elevated plasma asymmetric dimethyl-L-arginine levels are linked to endothelial progenitor cell depletion and carotid atherosclerosis in rheumatoid arthritis. Arthritis Rheum 2007;56:809–19. [9] Sari I, Kebapcilar L, Alacacioglu A, Bilgir O, Yildiz Y, Taylan A, et al. Increased levels of asymmetric dimethylarginine (ADMA) in patients with ankylosing spondylitis. Intern Med 2009;48:1363–8. [10] Sahin M, Arslan C, Naziroglu M, Tunc SE, Demirci M, Sutcu R, et al. Asymmetric dimethylarginine and nitric oxide levels as signs of endothelial dysfunction in Behcet's disease. Ann Clin Lab Sci 2006;36:449–54.
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Contents lists available at ScienceDirect
Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m
Asymmetric dimethylarginine (ADMA) levels are increased in patients with fibromyalgia: Correlation with tumor necrosis factor-α (TNF-α) and 8-iso-prostaglandin F2α (8-iso-PGF2α) G. Topal a,⁎, A. Donmez b, B.S. Uydes Doğan a, M. Kucur c, D. Taspinar Cengiz d, F.B. Berkoz b, N. Erdogan b a
Department of Pharmacology, Istanbul University, Faculty of Pharmacy, 34116, Beyazit, Istanbul, Turkey Department of Medical Ecology and Hydroclimatology, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey c Fikret Biyal Central Biochemistry Laboratory, Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey d Department of Statistics, Istanbul Commerce University, Faculty of Arts and Sciences, Istanbul, Turkey b
a r t i c l e
i n f o
Article history: Received 8 October 2010 Received in revised form 24 December 2010 Accepted 22 January 2011 Available online 1 February 2011 Keywords: Fibromyalgia ADMA TNF-α IL-6 8-iso-PGF2α
a b s t r a c t Objective: The aim of the study was to investigate serum levels of asymmetric dimethylarginine (ADMA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and plasma levels of 8-iso-prostaglandin F2α (8-iso-PGF2α) in patients with fibromyalgia. Design and method: Twenty-seven patients with fibromyalgia and twenty healthy controls were enrolled in this study. ADMA, TNF-α, IL-6 and 8-iso-PGF2α levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of ADMA and TNF-α and plasma levels 8-iso-PGF2α were significantly increased in patients with fibromyalgia compared to controls. However, no significant difference was observed in IL-6 levels between the two groups. ADMA concentrations were positively correlated with TNF-α and 8-iso-PGF2α levels in patients with fibromyalgia. Conclusion: This is the first study reporting that ADMA levels are significantly elevated in patients with fibromyalgia in association with increased 8-iso-PGF2α and TNF-α concentrations. Thereby, ADMA could be suggested as a reliable marker of endothelial dysfunction in patients with fibromyalgia. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Fibromyalgia (FM) is a form of nonarticular rheumatism characterized by chronic wide-spread pain, tender points, fatigue, nonrefreshing sleep and morning stiffness. The American College of Rheumatology 1990 Fibromyalgia Classification Criteria are based on a history of chronic widespread musculoskeletal pain for at least 3 months and more than 11 of 18 painful tender points with palpation [1]. FM is a very common rheumatic disorder among women, with a prevalence of 3.6% in Turkish population [2] which is similar to the prevalence of USA [3]. The etiology of FM is still unknown and the pathogenesis is not clearly understood. Of interest, an overlap is pointed out between FM and depression where a relationship between depression and coronary heart disease is very common. Thus, it is reasonable to propose a correlation between FM and coronary heart disease. In relation, an increase in the level of tenderness and occurrence of FM symptoms among patients with pathological findings on coronary angiography was documented [4]. In another study, left ventricular (LV) dysfunction was observed in FM
⁎ Corresponding author. Fax: +90 212 527 18 25. E-mail address: [email protected] (G. Topal).
patients with a high Fibromyalgia Impact Questionnaire (FIQ) score [5]. Hereby, the need of further investigations focusing on vascular and endothelial functions was signified in patients with FM. Favorably, endothelial dysfunction was reported very recently in patients with FM as assessed by the reduction in flow mediated dilation (FMD) of brachial artery [6]. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) and thus presumed as a marker of endothelial dysfunction. Elevated ADMA concentration has been recognized as an independent risk factor in atherosclerosis and related cardiovascular diseases [7]. Moreover, increased ADMA concentrations have been reported in various inflammatory rheumatic diseases, where endothelial dysfunction has been defined, including rheumatoid arthritis [8], ankylosing spondylitis [9] and Behçet's disease [10]. However, to the best of our knowledge, no information is evident in literature so far in relation to an association between ADMA levels and FM. It has previously been suggested that oxidative stress and proinflammatory cytokines play a significant role in the pathogenesis of FM [11,12]. Inflammation and oxidative stress are important determinants of the endothelial dysfunction by several mechanisms including the deterioration of nitric oxide (NO) bioavailability [7]. In relation, decreased NO production was suggested in FM in a previous
0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.01.011
G. Topal et al. / Clinical Biochemistry 44 (2011) 364–367
study [13]. Of note, an association between oxidative stress, proinflammatory cytokines and ADMA levels is demonstrated in various pathologies [14–16], whereas, no data are available in FM. Therefore, in this study, we aimed to examine serum levels of ADMA, a reliable marker of endothelial dysfunction, the proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as plasma levels of 8-iso-PGF2α, one of the important indicator of oxidative stress and lipid peroxidation, in patients with FM comparatively with control subjects. This study was also designed to examine the correlations among ADMA, TNF-α, IL-6 and 8-isoPGF2α levels in patients with FM. Patients and methods Patients
Table 2 Clinical characteristics of the patients with FM and controls.
Total cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL) Triglycerides (mg/dL) FIQ total Pain Fatigue Morning stiffness Anxiety Depression Tender points BDI Disease duration (year)
FM patients (n = 27)
Controls (19)
p-value
195.40 ± 33.35 51.67 ± 11.57 104.40 ± 17.73 100.80 ± 25.18 55.44 ± 15.98 5.88 ± 2.10 6.56 ± 2.36 4.60 ± 3.00 6.28 ± 2.85 4.72 ± 2.76 16.42 ± 1.77 10.76 ± 7.74 9.28 ± 5.06
184.4 ± 23.68 51.23 ± 11.26 117.4 ± 29.22 101.7 ± 42.69 27.57 ± 7.72 1.68 ± 1,24 3.05 ± 1.64 0.10 ± 0.31 2.78 ± 1.43 2.47 ± 1.71 2.11 ± 1.24 3.52 ± 2.91 –
0.326 0.925 0.198 0.951 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001 b 0.0001
HDL: high-density lipoprotein; LDL: low-density lipoprotein; FIQ: fibromyalgia impact questionnaire; BDI: Beck depression inventory. Values are given as mean ± SD.
Patients with FM were recruited from the musculoskeletal disorders outpatient clinic of Department of Medical Ecology and Hydroclimatology, Istanbul Medical Faculty. The study was performed on 27 patients satisfying the American College of Rheumatology Criteria for the classification of FM and 20 healthy controls matched for age. One healthy volunteer from control group recruited the study without any excuse. The patient and control groups were consisted of subjects at ages between 20 and 60, who did not have any other conditions in terms of chronic diseases and drug usage that may influence laboratory evaluation. The sociodemographic characteristics of the study groups are documented in Table 1. Laboratory tests used for the evaluation of patients and controls included complete blood count, erythrocyte sedimentation rate, glycaemia, serum lipid levels, hepatic and renal function tests, thyroid stimulant hormone level and X-ray. Exclusion criteria included the presence of any inflammatory autoimmune diseases, diabetes, renal disease, liver disease, known heart disease and pregnancy and use of any non-steroidal antiinflammatory drugs (NSAIDs) during a 15 days period before the collection of blood samples. Furthermore, our patients were allowed to use acetaminophen (max. 1 g/day) as an analgesic only when it is appropriate. The Institutional Review Board of the Istanbul University Faculty of Medicine approved the study plan and all patients and healthy controls voluntarily participated in the study and gave their written informed consent. Clinical evaluation FIQ was used for symptom measures and global evaluation of the patients. FIQ is a well-documented and widely-used measure in FM. FIQ is a ten-item questionnaire that measures physical functioning, work status, depression, anxiety, sleep, pain, stiffness, fatigue and well-being. [17]. FIQ is a widely recommended tool in FM research. The Turkish translation of the FIQ was found to be valid [18]. Biochemical analysis Venous blood was collected from each participant into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) or no anticoagulant. Immediately after delivery, serum or plasma was
Table 1 Sociodemographic characteristics of the study population.
Age (year) Sex (F/M) BMI (kg/m2) Education duration (year)
365
FM patients (n = 27)
Controls (19)
p-value
40.72 ± 10.58 23/2 24.52 ± 4.87 11.60 ± 4.74
40.00 ± 8.80 19/4 24.00 ± 3.18 14.68 ± 2.42
0.812 0.210 0.685 0.013
BMI: body mass index, F/M: female/male; values are given as mean ± SD.
isolated by centrifugation at 2500g for 10 min at 4 °C and stored at −80 °C until the analyses were performed. Plasma 8-iso-PGF2α levels and serum TNF-α, IL-6 and ADMA levels were analyzed by using commercial enzyme-linked immunosorbent assay (ELISA) kit for 8-iso-PGF2α, TNF-α, IL-6 (Cayman Chemical, Ann Arbor, MI, USA) and for ADMA (DLD, Diagnostika GmbH, Hamburg, Germany) according to the manufacturer's instructions. Intra-assay coefficient of variation was 5.7%, 10.6%, 15.4%, 14.9% and inter-assay coefficient of variation was 10.3%, 18.3%, 10.4% and 23.1% for ADMA, 8-isoPGF2α, TNF-α and IL-6, respectively. All samples were assayed in duplicate and the concentration was calculated from a standard curve of the corresponding recombinant proteins. Serum lipid levels were measured according to standard procedures by the local clinical laboratory. Statistical analysis All statistical analyses were performed using Statistical Package for Social Sciences software (SPSS, Chicago, IL, USA) version 18.0 for windows “n” denotes the number of FM and control subjects. All values are given as mean ± standard deviation (SD). Differences between the FM and control groups were evaluated by using Student's unpaired t-test. For determination of correlation between results, the Pearson's correlation coefficient was performed. Furthermore, the partial correlation analyses were fitted to evaluate the extend to which the associations between variables was altered after adjusting for age, gender and body mass index (BMI). p b 0.05 was considered statistically significant. Results Table 1 presents the sociodemographic characteristics of the study population. No significant differences were found between the groups with regard to age, gender and BMI. But, the controls had longer education duration than patients with FM. Clinical characteristics of the patients and control subjects are presented on Table 2. The main symptoms of FM are found to be pain, fatigue, and morning stiffness. Anxiety and depression are also frequently observed in patients with FM. In addition, no significant difference was determined in the lipid profile of FM and control groups (Table 2). Serum ADMA (p = 0.039) and TNF-α (p = 0.008) levels and plasma 8-iso-PGF2α levels (p = 0.001) were significantly increased in patients with FM compared to controls (Table 3). However, no significant difference was observed in terms of serum IL-6 levels between two groups (p = 0.992). According to the results of unadjusted correlation analysis, ADMA concentrations were found to be correlated significantly with both TNF-α and 8-iso-PGF2α levels (r = 0.549, p = 0.007 and r = 0.463,
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Table 3 Biochemical parameters in patients with FM and controls.
ADMA (μmol/L) TNF-α (pg/mL) IL-6 (pg/mL) 8-iso-PGF2α (pg/mL)
FM patients (n = 27)
Controls (n = 19)
p-value
0.78 ± 0.31 21.37 ± 15.35 10.33 ± 8.55 165.37 ± 98.71
0.58 ± 0.30 10.32 ± 5.86 10.29 ± 11.47 72.78 ± 59.21
0.039 0.008 0.992 0.001
Values are mean ± SD.
Table 5 Adjusted correlation of ADMA versus TNF-α, 8-iso-PGF2α and IL-6 levels in patients with FM and controls.
TNF-α 8-iso-PGF2α IL-6
FM patients (27)
Control (19)
r
p
r
p
0.613 0.612 0.495
0.005 0.004 0.061
−0.030 0.167 −0.057
0.916 0.538 0.853
Partial correlation analyses were used to adjust for age, gender and BMI.
p = 0.015, respectively) in patients with FM (Table 4). After adjustment for age, gender and BMI with partial correlation, the observed associations between these variables remained significant (Table 5). In addition, no significant correlation was observed between ADMA and IL-6 concentrations in both unadjusted and adjusted correlation analysis in patients with FM. Whereas, in control subjects, no significative correlation was observed between the variables in either the adjusted or unadjusted analyses (Tables 4 and 5). Discussion In this study, we demonstrated that serum ADMA and TNF-α levels and plasma 8-iso-PGF2α levels were significantly elevated in patients with FM while IL-6 levels were unaltered. Moreover, ADMA levels were significantly correlated with TNF-α and 8-iso-PGF2α levels in these patients. ADMA, an endogenous inhibitor of NO synthase (NOS), is a relevant and independent risk factor for cardiovascular diseases. Endothelial dysfunction is commonly observed in cardiovascular diseases which is mainly attributed to oxidative stress, dyslipidemia, genetic factors and other causes. There is a growing evidence indicating that increased levels of ADMA may also contribute to endothelial dysfunction [7,19]. Moreover, recent studies suggest that ADMA is also a potential candidate to enhance vascular complications in patients with inflammatory rheumatic diseases including rheumatoid arthritis [8], ankylosing spondylitis [9] and Behçet's disease [10]. In the current study, we have reported for the first time that serum ADMA levels are elevated in patients with FM which may reflect the presence of endothelial dysfunction. Indeed, our findings presented an explanation for the previous hypothesis of endothelial dysfunction and decrease of NO production in FM [13]. Thus, enhanced ADMA generation might be associated with the risk of future cardiovascular events in these patients. Of interest, an association between FM and coronary heart diseases has been emphasized in the recent studies. Firstly, Ablin J.N. et al. demonstrated an increase in the levels of tenderness and in occurrence of FM symptoms among patients with pathological findings on coronary angiography [4]. Then, in another study, left ventricular (LV) dysfunction was observed in FM patients with a high FIQ score [5]. Finally, in a latest study, decreased FMD of branchial artery was reported in patients with FM which supports the role of endothelial dysfunction [6]. Hence, in the present study, increased ADMA levels were defined in patients with FM which provide a further evidence in the relationship between endothelial dysfunction, cardiovascular disorders, and FM. However, additional research is required to demonstrate the association between ADMA levels and flow-mediated vasodilation directly in patients with FM.
Table 4 Correlation of ADMA versus TNF-α, 8-iso-PGF2α and IL-6 levels in patients with FM and controls. FM patients (27)
TNF-α 8-iso-PGF2α IL-6
Control (19)
r Pearson
p
r Pearson
p
0.549 0.463 0.126
0.007 0.015 0.607
0.049 0.153 0.220
0.857 0.546 0.431
Proinflammatory cytokines might play a regulatory role in ADMA metabolism. In an in vitro study carried on human umbilical vein endothelial cells, TNF-α was shown to increase the accumulation of ADMA whereas decreased dimethylarginine dimethyl-aminohydrolase (DDAH) activity which is responsible for the metabolism of ADMA [20]. Furthermore, in patients with acute liver failure, elevated plasma concentration of ADMA was shown to correlate with the concentrations of TNF-α and IL-6 possibly through the regulation of ADMA metabolism by these cytokines [16]. The activation and regulation of pro-inflammatory cytokine pattern are implied in a variety of disease states such as, rheumatoid arthritis, ankylosing spondylitis [21]. Moreover, the association between FM and proinflammatory cytokines has been evaluated in some of recent studies [11,22]. In support, in the current study, we have found elevated serum levels of TNF-α. However, the other proinflammatory cytokine IL-6 levels were found indifferent in patients with FM compared to controls. These data are consistent with other studies in the literature reporting similarity in serum or plasma levels of IL-6 [11,23,24] whereas an increase in the levels of TNF-α [11,22] in patients with FM compared to healthy controls. Lastly, our results indicating a positive correlation between increased ADMA and TNF-α levels in patients with FM, which was significant even after adjustments for age, gender and BMI, supported the hypothesis of an association between a cytokinedriven inflammatory response and ADMA levels. F2-isoprostanes including 8-iso-PGF2α are currently considered the best available biomarkers of oxidative stress and lipid peroxidation in vivo [25]. Elevated 8-iso-PGF2α levels have been found in different inflammatory conditions including rheumatic diseases as well [26]. Additionally, increased levels of plasma 8-iso-PGF2α have been demonstrated in chronic fatigue syndrome which displays similar symptoms with FM [27]. In our study, it is clearly evident that plasma 8-iso-PGF2α levels are significantly higher in patients with FM than controls, indicating an enhanced oxidative stress and lipid peroxidation in these patients. Actually, this finding is in agreement with previous studies that have linked oxidative stress and lipid peroxidation to FM pathogenesis [12,28–31]. In fact, this is the first study indicating the role of 8-iso-PGF2α, possibly as an oxidant candidate, in FM. In contrary to our findings, Chung et al. did not demonstrate an alteration in urinary excretion of 8-iso-PGF2α in FM patients [32]. This discrepancy may be related to the differences in test samples or the methodology used in the analysis of 8-iso-PGF2α. Oxidative stress is now considered as a crucial factor in ADMA accumulation [33]. In relation, the increased ADMA generation associated with 8-iso-PGF2α levels was very recently reported in inflammatory bowel diseases [14]. Hence, in the present study, this relevance is furtherly supported by the positive correlation verified between ADMA and 8-iso-PGF2α levels in patients with FM which remained significant even after adjustments for age, gender and BMI. This study had some limitations that should be considered. Firstly, the number of patients is limited and therefore, this issue requires further studies with much larger subject population to give greater confidence in statistical correlations. Secondly, we did not assess vascular endothelial function in patients with FM. Further studies such as the measurement of flow mediated vasodilation are
G. Topal et al. / Clinical Biochemistry 44 (2011) 364–367
appropriate to validate the correlation between increased ADMA levels and endothelial dysfunction in FM. In conclusion, present study primarily shows that in patients with FM an enhancement is prominent in serum ADMA levels which is positively correlated with increased 8-iso-PGF2α and TNF-α concentrations. These results suggest that endothelial dysfunction might play a prominent role in the pathophysiology of FM. In consequence, patients with FM might be more prone to cardiovascular disorders. Hence, current findings strengthen the earlier hypothesis of Ablin et al. [4] and Cho et al. [5] on the association between coronary heart diseases and FM as well as the latest report by Cho et al. [6] on the presence of endothelial dysfunction assessed by a decrease in FMD of branchial artery in patients with FM. Lastly, our study strongly indicates the necessity for further investigations that reveal vascular functional properties particularly in the follow up period in patients with FM. Conflict of interest The authors declare that they have no competing interests. Acknowledgments The authors thank Ms. Nevin Bezgal and Ms. Nuray Çetin for excellent technical assistance. References [1] Wolfe F, Smythe HA, Yunus MB, Bennett RM, Bombardier C, Goldenberg DL, et al. The American College of Rheumatology 1990 Criteria for the classification of fibromyalgia. Report of the Multicenter Criteria Committee. Arthritis Rheum 1990;33:160–72. [2] Topbas M, Cakirbay H, Gulec H, Akgol E, Ak I, Can G. The prevalence of fibromyalgia in women aged 20–64 in Turkey. Scand J Rheumatol 2005;34:140–4. [3] Wolfe F, Ross K, Anderson J, Russell IJ, Hebert L. The prevalence and characteristics of fibromyalgia in the general population. Arthritis Rheum 1995;38:19–28. [4] Ablin JN, Beilinson N, Aloush V, Elkayam O, Finkelstein A. Association between fibromyalgia and coronary heart disease and coronary catheterization. Clin Cardiol 2009;32(6):E7–E11. [5] Cho KI, Lee JH, Lee HG, Kim SM, Kim TI. Assessment of myocardial function in patients with fibromyalgia and relationship to chronic emotional and physical stress. Korean Circ J 2010;40:74–80. [6] Cho KI, Lee JH, Kim SM, Lee HG, Kim TI. Assessment of endothelial function in patients with fibromyalgia-cardiac ultrasound study. Clin Rheumatol 2010 Oct 19. [Epub ahead of print]. [7] Boger RH. Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond. Ann Med 2006;38:126–36. [8] Surdacki A, Martens-Lobenhoffer J, Wloch A, Marewicz E, Rakowski T, WieczorekSurdacka E, et al. Elevated plasma asymmetric dimethyl-L-arginine levels are linked to endothelial progenitor cell depletion and carotid atherosclerosis in rheumatoid arthritis. Arthritis Rheum 2007;56:809–19. [9] Sari I, Kebapcilar L, Alacacioglu A, Bilgir O, Yildiz Y, Taylan A, et al. Increased levels of asymmetric dimethylarginine (ADMA) in patients with ankylosing spondylitis. Intern Med 2009;48:1363–8. [10] Sahin M, Arslan C, Naziroglu M, Tunc SE, Demirci M, Sutcu R, et al. Asymmetric dimethylarginine and nitric oxide levels as signs of endothelial dysfunction in Behcet's disease. Ann Clin Lab Sci 2006;36:449–54.
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